It is anticipated that a monoclonal antibody exhibiting high selectivity to a specific antigen will be developed as an antibody drug, and in particular, the development of antibody drugs targeting cancer cells has progressed. In order to apply a medicament comprising a monoclonal antibody as an active ingredient to the treatment for humans, administration of a human antibody with a small amount of foreign antigen is most ideal from the viewpoint of the avoidance of rejection. Hence, a large number of chimeric antibodies and humanized antibodies are developed.
In general, chimeric antibodies or humanized antibodies used for the treatment of humans are produced by immunizing mice or other animals with antigens several times, then fusing cells in the spleen or lymph nodes with myeloma cells to form hybridomas, and then applying a recombination technique to mouse IgG antibodies produced from the hybridomas. However, it takes much time to use an individual animal such as a mouse or to select hybridomas that generate antibodies exhibiting a high affinity. Moreover, it is necessary to confirm the activity of an antibody obtained by a recombination technique. Thus, it takes time to produce an antibody of interest by the aforementioned method, and further, the effects of the produced antibody cannot be secured until it is administered to a human. Furthermore, when an immunogen exhibits toxicity to individual, immunization to an individual is difficult. Further, when a protein antigen that is highly preserved among animal species is used as an antigen, an antibody is hardly generated due to immunological tolerance.
B cells are immune cells that are derived from bone marrow, have B cell receptors (BCR) on the surface thereof, and produce antibodies. Such B cells are generated from hematopoietic stem cells, and the cells are differentiated into B cells through the stage of pro-B cells and pre-B cells. It has been known that the B cells producing antibodies exhibiting a high affinity for antigens are selected in a germinal center. However, this selection mechanism has not yet been elucidated. If such B cells producing antibodies exhibiting a high affinity for specific antigens were allowed to artificially proliferate and could be then concentrated, monoclonal antibodies exhibiting a high affinity for specific antigens could be produced in a shorter time than previous techniques.
As a method for proliferation of B cells, there has been known a method of culturing B cells in the presence of a CD40 ligand (CD40L) and cytokine such as interleukin (IL)-4 (for example, Patent Literature 1 and Non Patent Literature 1).
Moreover, the germinal center is a histological structure formed as a result that naive B cells, which had been uncontacted with antigens, have been contacted with antigens, and have thereby proliferated. It has been known that class switching or somatic hypermutation would take place in the B cells in the germinal center. For example, Non Patent Literature 2 discloses that, when splenic B cells are cultured together with IL-4 and an anti-μHc antibody in the presence of fibroblasts in which BAFF and CD40L have been allowed to express simultaneously, almost all of the B cells exhibit a germinal center-like phenotype, and as a result, about a half thereof is class-switched to IgG1, and that if IL-4 is then replaced with IL-21, IgE-positive cells increase.
Patent Literature 2 discloses a method for producing an antigen-specific B cell population comprising IgG-positive B cells specific to specific antigens, wherein the method comprises culturing IgG-positive B cells together with the specific antigens in the presence of IL-21, while giving stimulation mediated by CD40, a BAFF receptor and Fas to the cells, and then selecting antigen-specific B cells specific to the specific antigens from the cultured cells, so as to obtain an antigen-specific B cell population comprising IgG-positive B cells specific to the specific antigens.
Patent Literature 3 discloses a method of stabilizing a B cell line, comprising a step of obtaining B cells, a step of increasing the expression of BCL-6 in the B cells, and a step of maintaining the cells in an environment in which the cells can be replicated.
Patent Literature 4 discloses a method of influencing the stability of antibody-producing cells, comprising a stage of directly or indirectly influencing the amounts of expression products of BCL6 and/or Blimp-1 in the antibody-producing cells.
Patent Literature 5 discloses a non-human animal that is characterized in that the expression of a bach2 gene is artificially suppressed, and that IgM and/or IgM-producing hybridomas can be produced at a high frequency by using the aforementioned animal.